U.S. patent number 10,301,277 [Application Number 15/759,119] was granted by the patent office on 2019-05-28 for substituted ureas and methods of treating mental illness using the same.
This patent grant is currently assigned to SHANGHAI JINGXIN BIOMEDICAL CO., LTD., SHANGYU JINGXIN PHARMACEUTICAL CO., LTD., ZHEJIANG JINGXIN PHARMACEUTICAL CO., LTD.. The grantee listed for this patent is SHANGHAI JINGXIN BIOMEDICAL CO., LTD., SHANGYU JINGXIN PHARMACEUTICAL CO., LTD., ZHEJIANG JINGXIN PHARMACEUTICAL CO., LTD.. Invention is credited to Yue Huang, Fei Zheng.
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United States Patent |
10,301,277 |
Huang , et al. |
May 28, 2019 |
Substituted ureas and methods of treating mental illness using the
same
Abstract
A cyclohexane derivative as shown by formula IB or a
stereoisomer or a salt thereof, has a high affinity for D.sub.3
receptors and 5-hydroxytryptamine, has a lower affinity for D.sub.2
receptors, shows a high selectivity for D.sub.3/D.sub.2 receptors,
and can be used as a therapeutic drug against neuropsychiatric
diseases. ##STR00001## wherein; X is N or CH; R is ##STR00002## R
is optionally substituted with one or more substituents selected
from the group consisting of F, Cl, Br, I and C.sub.1-C.sub.6
alkyl; and the C.sub.1-C.sub.6 alkyl is optionally substituted with
one or more substituents selected from the group consisting of F,
Cl, Br, and I.
Inventors: |
Huang; Yue (Shanghai,
CN), Zheng; Fei (Shanghai, CN) |
Applicant: |
Name |
City |
State |
Country |
Type |
ZHEJIANG JINGXIN PHARMACEUTICAL CO., LTD.
SHANGHAI JINGXIN BIOMEDICAL CO., LTD.
SHANGYU JINGXIN PHARMACEUTICAL CO., LTD. |
Zhejiang
Pudong Shanghai
Shangyu, Zhejiang |
N/A
N/A
N/A |
CN
CN
CN |
|
|
Assignee: |
ZHEJIANG JINGXIN PHARMACEUTICAL
CO., LTD. (CN)
SHANGHAI JINGXIN BIOMEDICAL CO., LTD. (CN)
SHANGYU JINGXIN PHARMACEUTICAL CO., LTD.
(CN)
|
Family
ID: |
58343473 |
Appl.
No.: |
15/759,119 |
Filed: |
September 14, 2016 |
PCT
Filed: |
September 14, 2016 |
PCT No.: |
PCT/CN2016/098953 |
371(c)(1),(2),(4) Date: |
March 09, 2018 |
PCT
Pub. No.: |
WO2017/045599 |
PCT
Pub. Date: |
March 23, 2017 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20180297975 A1 |
Oct 18, 2018 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D
261/20 (20130101); A61K 9/2059 (20130101); C07D
333/72 (20130101); C07D 413/04 (20130101); A61P
25/18 (20180101); A61K 31/496 (20130101); C07D
417/04 (20130101); A61K 9/2018 (20130101); C07D
333/54 (20130101); A61K 9/2054 (20130101); A61K
31/454 (20130101); C07D 409/04 (20130101); C07D
275/04 (20130101); A61K 9/2013 (20130101) |
Current International
Class: |
C07C
275/26 (20060101); A61P 25/18 (20060101); A61K
9/20 (20060101); A61K 31/454 (20060101); A61K
31/496 (20060101); C07D 261/20 (20060101); C07D
409/04 (20060101); C07D 417/04 (20060101); C07D
275/04 (20060101); C07D 333/54 (20060101); C07D
333/72 (20060101); C07D 413/04 (20060101) |
Field of
Search: |
;564/57 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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101107236 |
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Jan 2008 |
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CN |
|
101511805 |
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Aug 2009 |
|
CN |
|
101778844 |
|
Jul 2010 |
|
CN |
|
102159557 |
|
Aug 2011 |
|
CN |
|
102164914 |
|
Aug 2011 |
|
CN |
|
102164927 |
|
Aug 2011 |
|
CN |
|
102958936 |
|
Mar 2013 |
|
CN |
|
103130737 |
|
Jun 2013 |
|
CN |
|
104140421 |
|
Nov 2014 |
|
CN |
|
Other References
WIPO, International Search Report for International Application No.
PCT/CN2016/098953, dated Dec. 13, 2016 (8 pages). cited by
applicant.
|
Primary Examiner: Willis; Douglas M
Attorney, Agent or Firm: Haynes and Boone, LLP
Claims
What is claimed is:
1. A compound shown by the formula IB: ##STR00038## or a
pharmaceutically acceptable salt or stereoisomer thereof, wherein:
X is N or CH; and R is ##STR00039## where R is optionally
substituted with one or more substituents selected from the group
consisting of F, Cl, Br, I and C.sub.1-C.sub.6 alkyl; and further
where the C.sub.1-C.sub.6 alkyl is optionally substituted with one
or more substituents selected from the group consisting of F, Cl,
Br and I.
2. The compound according to claim 1, or a pharmaceutically
acceptable salt or stereoisomer thereof, wherein R is substituted
with one C.sub.1-C.sub.4 alkyl, where the C.sub.1-C.sub.4 alkyl is
substituted with one or more F.
3. The compound according to claim 1, or a pharmaceutically
acceptable salt or stereoisomer thereof, wherein the
pharmaceutically acceptable salt is an acid addition salt formed
with an acid selected from the group consisting of hydrochloric
acid, sulfuric acid, nitric acid, phosphoric acid, formic acid,
acetic acid, oxalic acid, malonic acid, maleic acid, fumaric acid,
succinic acid and benzoic acid.
4. The compound according to claim 1, or a pharmaceutically
acceptable salt or stereoisomer thereof, wherein the stereoisomer
is the cis-stereoisomer or the trans-stereoisomer.
5. The compound according to claim 1, or a pharmaceutically
acceptable salt or stereoisomer thereof, wherein the compound is
selected from the group consisting of:
N'-[trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea, compound 1;
N'-[trans-4-[2-[7-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea, compound 2;
N'-[trans-4-[2-[4-(benzo[c]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea, compound 3;
N'-[trans-4-[2-[4-(6-fluorobenzo[d]isoxazol)-3-piperazinyl]ethyl]cyclohex-
yl]-N,N-dimethylurea, compound 5;
N'-[trans-4-[2-[4-(3-chlorobenzo[d]isoxazol)-6-piperazinyl]ethyl]cyclohex-
yl]-N,N-dimethylurea, compound 6;
N'-[trans-4-[2-[4-(6-fluorobenzo[d]isoxazol)-3-piperidinyl]ethyl]cyclohex-
yl]-N,N-dimethylurea, compound 7
N'-[trans-4-[2-[4-(benzo[b]thiophene)-7-piperidinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea, compound 8;
N'-[trans-4-[2-[7-(benzo[b]thiophene)-7-piperidinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea, compound 9;
N'-[trans-4-[2-[4-(benzo[c]thiophene)-7-piperidinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea, compound 10;
N'-[trans-4-[2-[4-(benzo[d]isothiazolyl)-3-piperidinyl]ethyl]cyclohexyl]--
N,N-dimethylurea, compound 11;
N'-[trans-4-[2-[4-(3-chlorobenzo[d]isoxazol)-6-piperidinyl]ethyl]cyclohex-
yl]-N,N-dimethylurea, compound 12;
N'-[trans-4-[2-[4-(3-methylbenzo[d]isoxazol)-6-piperazinyl]ethyl]cyclohex-
yl]-N,N-dimethylurea, compound 13; and
N'-[trans-4-[2-[4-(6-methylbenzo[d]isoxazol)-4-piperazinyl]ethyl]cyclohex-
yl]-N,N-dimethylurea, compound 14.
6. A pharmaceutical composition comprising as an active ingredient
a compound according to claim 1, or a pharmaceutically acceptable
salt or stereoisomer thereof, and a pharmaceutically acceptable
excipient.
7. The pharmaceutical composition according to claim 6, wherein the
pharmaceutical composition is a solid or a liquid preparation for
oral, gastrointestinal, buccal, sublingual, nasal, rectal or
transdermal administration.
8. The pharmaceutical composition according to claim 7, wherein the
pharmaceutical composition is a solid tablet.
9. The pharmaceutical composition according to claim 8, wherein the
compound in the solid tablet is
N'-[trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea.
10. The pharmaceutical composition according to claim 7, wherein
the pharmaceutical composition is a suspension.
11. The pharmaceutical composition according to claim 10, wherein
the compound in the suspension is N'-[trans-4-[2-[4-(benzo
[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-dimethylurea.
12. A method for inhibiting dopamine D2 receptor binding activity
in a patient, comprising administering to a patient in need thereof
a therapeutically effective amount of the pharmaceutical
composition according to claim 6.
13. The method according to claim 12, wherein the patient suffers
from one or more disorders selected from the group consisting of a
phobia, a mental disorder, a mood disorder, a cognitive disorder
and an obsessive-compulsive disorder.
14. The method according to claim 13, wherein the mental disorder,
mood disorder or cognitive disorder is selected from the group
consisting of mental confusion, schizophrenia, depression, anxiety,
dysphrenia and a bipolar disorder.
15. A method for inhibiting 5-hydroxytryptamine 1A receptor binding
activity in a patient, comprising administering to a patient in
need thereof a therapeutically effective amount of the
pharmaceutical composition according to claim 6.
16. The method according to claim 15, wherein the patient suffers
from one or more disorders selected from the group consisting of a
phobia, a mental disorder, a mood disorder, a cognitive disorder
and an obsessive-compulsive disorder.
17. The method according to claim 16, wherein the mental disorder,
mood disorder or cognitive disorder is selected from the group
consisting of mental confusion, schizophrenia, depression, anxiety,
dysphrenia and a bipolar disorder.
18. A process for preparing a compound according to claim 1 shown
by the formula IB: ##STR00040## wherein the process comprises:
reacting a compound of the formula II: ##STR00041## wherein: X is N
or CH: and ##STR00042## R is where R is optionally substituted with
one or more substituents selected from the group consisting of F,
Cl, Br, I and C.sub.1-C.sub.6 alkyl; and further where the
C.sub.1-C.sub.6 alkyl is optionally substituted with one or more
substituents selected from the group consisting of F, Cl, Br and I;
with a compound of the formula III: ##STR00043## in the presence of
a base selected from the group consisting of triethylamine,
diisopropylethylamine, pyridine, sodium carbonate, potassium
carbonate, sodium bicarbonate and potassium bicarbonate, to provide
the compound according to claim 1 shown by the formula IB
above.
19. A process for preparing the pharmaceutical composition
according to claim 6, wherein the process comprises admixing a
pharmaceutically acceptable excipient with the compound according
to claim 1, or a pharmaceutically acceptable salt or stereoisomer
thereof.
20. A compound shown by the formula I: ##STR00044## or a
pharmaceutically acceptable salt or stereoisomer thereof, wherein:
R is ##STR00045## where R is optionally substituted with one or
more substituents selected from the group consisting of F, Cl, Br,
I and C.sub.1-C.sub.6 alkyl; and further where the C.sub.1-C.sub.6
alkyl is optionally substituted with one or more substituents
selected from the group consisting of F, Cl, Br and I.
21. The compound according to claim 20, or a pharmaceutically
acceptable salt or stereoisomer thereof, wherein R is substituted
with one C.sub.1-C.sub.4 alkyl, where the C.sub.1-C.sub.4 alkyl is
substituted with one or more F.
22. The compound according to claim 20, or a pharmaceutically
acceptable salt or stereoisomer thereof, wherein the
pharmaceutically acceptable salt is an acid addition salt formed
with an acid selected from the group consisting of hydrochloric
acid, sulfuric acid, nitric acid, phosphoric acid, formic acid,
acetic acid, oxalic acid, malonic acid, maleic acid, fumaric acid,
succinic acid and benzoic acid.
23. The compound according to claim 20, or a pharmaceutically
acceptable salt or stereoisomer thereof, wherein the stereoisomer
is the trans-stereoisomer.
24. The compound according to claim 20, or a pharmaceutically
acceptable salt or stereoisomer thereof, wherein the compound is
selected from the group consisting of:
N'-[trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea, compound 1;
N'-[trans-4-[2-[7-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea, compound 2;
N'-[trans-4-[2-[4-(benzo[c]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea, compound 3;
N'-[trans-4-[2-[4-(6-fluorobenzo[d]isoxazol)-3-piperazinyl]ethyl]cyclohex-
yl]-N,N-dimethylurea, compound 5;
N'-[trans-4-[2-[4-(3-chlorobenzo[d]isoxazol)-6-piperazinyl]ethyl]cyclohex-
yl]-N,N-dimethylurea, compound 6;
N'-[trans-4-[2-[4-(3-methylbenzo[d]isoxazol)-6-piperazinyl]ethyl]cyclohex-
yl]-N,N-dimethylurea, compound 13; and
N'-[trans-4-[2-[4-(6-methylbenzo[d]isoxazol)-4-piperazinyl]ethyl]cyclohex-
yl]-N,N-dimethylurea, compound 14.
25. A pharmaceutical composition comprising as an active ingredient
a compound according to claim 20, or a pharmaceutically acceptable
salt or stereoisomer thereof, and a pharmaceutically acceptable
excipient.
26. A process for preparing a compound according to claim 20 shown
by the formula I: ##STR00046## wherein the process comprises:
reacting a compound of the formula II: ##STR00047## wherein: R is
##STR00048## where R is optionally substituted with one or more
substituents selected from the group consisting of F, Cl, Br, I and
C.sub.1-C.sub.6 alkyl; and further where the C.sub.1-C.sub.6 alkyl
is optionally substituted with one or more substituents selected
from the group consisting of F, Cl, Br and I; with a compound of
the formula III: ##STR00049## in the presence of a base selected
from the group consisting of triethylamine, diisopropylethylamine,
pyridine, sodium carbonate, potassium carbonate, sodium bicarbonate
and potassium bicarbonate, to provide the compound according to
claim 20 shown by the formula I above.
27. A process for preparing the pharmaceutical composition
according to claim 25, wherein the process comprises admixing a
pharmaceutically acceptable excipient with the compound according
to claim 20, or a pharmaceutically acceptable salt or stereoisomer
thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national stage entry of PCT/CN2016/098953
filed Sep. 14, 2016, which claims priority to Chinese Patent
Application No. 201510582267.0 filed Sep. 15, 2015 and Chinese
Patent Application No. 201610643389.0 filed Aug. 8, 2016, the
contents of each of which are incorporated herein in their entirety
by express reference thereto.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable
THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT
Not Applicable
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT
DISC
Not Applicable
BACKGROUND OF THE INVENTION
Field of the Invention
The present application relates to medicinal chemistry, in
particular to cyclohexane compounds or stereoisomers or salts
thereof, and more particularly to cyclohexane derivatives of
formula IB or formula I, or stereoisomers or salts thereof, and
preparation and use thereof.
Description of Related Art
Mental disorders have been the diseases that seriously affect human
health with the rapid social development, the increased pace and
stress from lives, which leads to bad consequences for the patients
and their families. Suicide, deficiency of medical care and high
risk of complications (for example, malnutrition, insufficient
exercises, obesity and smoking) are contributors to shortened
average life expectancy of patients. Many studies have shown that
mental disorders are associated with various neurotransmitters and
receptor dysfunction in the central nervous system; for example,
monoamine neurotransmitters in brain, especially dopamine (DA)
system and 5-hydroxytryptamine (5-HT) system are closely related to
the normal mental activities. Dysfunction of DA and 5-HT systems
can lead to a variety of neuropsychiatric diseases, such as
schizophrenia, depression, neuropathic pain, mania, anxiety and
Parkinson's disease.
The Patent WO 9967206 A1 discloses an application of a cyclohexane
derivative in the treatment of pain diseases, but is silent about
the application in mental diseases, especially for dopamine
D.sub.2/D.sub.3 receptors.
The Patent CN 1829703 A discloses an application of a cyclohexane
derivative having (thio) carbamoyl side chain in the modulation of
dopamine receptor-related disorders, in which the D.sub.2/D.sub.3
antagonist and 5-HT.sub.1A partial agonist Cariprazine
(Cariprazine, RGH-188) jointly developed by Forest Laboratories and
Gedeon Richter for the treatment of schizophrenia, mania and
depression have now passed clinical trials and entered the
registration and approval stage. Cariprazine has a formula as shown
below, and has affinities (Ki values) of 0.72 nmol, 0.08 nmol and
3.42 nmol for D.sub.2/D.sub.3 receptors and 5-HT.sub.1A,
respectively, i.e. it does have a certain selectivity to D2/D3
receptor, but still not ideal. It is therefore possible that such
drug clinically has less chance (nearly 5% probability at a dose of
3 mg) on the occurrence of the cathisophobia, extrapyramidal
reaction as these side effects are associated with excessive
blocking of the D.sub.2 receptor.
##STR00003##
In light of the above problems, Cariprazine is further structurally
modified in Patent CN 103130737 A so as to achieve higher
selectivity to the D.sub.3 receptor.
However, given the various causes for mental diseases, there
remains a need for the development of the medicaments to meet the
requirement from the treatment of mental diseases, although the
compounds as described above function well against
schizophrenia.
BRIEF SUMMARY OF THE INVENTION
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
Not Applicable
The technical problem to be solved in the present invention is to
overcome the above shortcomings, to study, design and improve the
cyclohexane derivative structure. The present application provides
a cyclohexane derivative of formula IB and formula I, or a
stereoisomer or a salt thereof, which produces D.sub.2/D.sub.3
antagonism and 5-hydroxytryptamine absorption inhibition, as well
as anti-schizophrenia effect, thus increasing the spectrum of
mental illness treatment and reducing side effects.
The present invention provides a cyclohexane derivative of formula
IB, or a stereoisomer or salt thereof:
##STR00004## wherein X is N or C; R is
##STR00005## and R group is optionally substituted with one or more
substituents selected from halogen, substituted or unsubstituted
C.sub.1-C.sub.6 alkyl; the halogen is selected from one or more of
F, Cl, Br or I; the substituted or unsubstituted C.sub.1-C.sub.6
alkyl is selected from a substituted or unsubstituted
C.sub.1-C.sub.4 alkyl, for example methyl, ethyl, propyl or butyl;
the substituent is halogen, for example one or more of F, Cl, Br or
I; and the substituted C.sub.1-C.sub.4 alkyl is preferably
trifluoromethyl.
Preferably, the present invention provides a cyclohexane derivative
of formula I, or a stereoisomer or salt thereof:
##STR00006## wherein R is
##STR00007## and R group is optionally substituted with one or more
substituents selected from halogen, substituted or unsubstituted
C.sub.1-C.sub.6 alkyl; the halogen is selected from one or more of
F, Cl, Br or I; the substituted or unsubstituted C.sub.1-C.sub.6
alkyl is selected from a substituted or unsubstituted
C.sub.1-C.sub.4 alkyl, for example methyl, ethyl, propyl or butyl;
the substituent is halogen, for example one or more of F, Cl, Br or
I; and the substituted C.sub.1-C.sub.4 alkyl is preferably
trifluoromethyl.
The stereoisomer of the cyclohexane derivative of the present
invention is a cis-stereoisomer or a trans-stereoisomer, preferably
a trans-stereoisomer.
The salt of the cyclohexane derivative of the present invention is
formed from a cyclohexane derivative with an acid which is an
organic or inorganic acid, wherein the inorganic acid is selected
from hydrochloric acid, sulfuric acid, nitric acid or phosphoric
acid; the organic acid is selected from formic acid, acetic acid,
oxalic acid, malonic acid, maleic acid, fumaric acid, succinic acid
or benzoic acid; and other physiologically acceptable salt.
Preferably, the cyclohexane derivative or a stereoisomer or salt
thereof is selected from the following compounds or salts thereof:
N'-[trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea (compound 1);
##STR00008##
N'-[trans-4-[2-[7-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea (compound 2);
##STR00009##
N'-[trans-4-[2-[4-(benzo[c]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea (compound 3);
##STR00010##
N'-[trans-4-[2-[4-(benzo[d]isothiazolyl)-3-piperazinyl]ethyl]cyclohexyl]--
N,N-dimethylurea (compound 4);
##STR00011##
N'-[trans-4-[2-[4-(6-fluoro-benzo[d]isoxazol)-3-piperazinyl]ethyl]cyclohe-
xyl]-N,N-dimethylurea (compound 5);
##STR00012##
N'-[trans-4-[2-[4-(3-chloro-benzo[d]isoxazol)-6-piperazinyl]ethyl]cyclohe-
xyl]-N,N-dimethylurea (compound 6);
##STR00013##
N'-[trans-4-[2-[4-(6-fluoro-benzo[d]isoxazol)-3-piperidyl]ethyl]cyclohexy-
l]-N,N-dimethylurea (compound 7);
##STR00014##
N'-[trans-4-[2-[4-(benzo[b]thiophene)-7-piperidinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea (compound 8);
##STR00015##
N'-[trans-4-[2-[7-(benzo[b]thiophene)-7-piperidinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea (compound 9);
##STR00016##
N'-[trans-4-[2-[4-(benzo[c]thiophene)-7-piperidinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea (compound 10);
##STR00017##
N'-[trans-4-[2-[4-(benzo[d]isothiazolyl)-3-piperidinyl]ethyl]cyclohexyl]--
N,N-dimethylurea (compound 11);
##STR00018##
N'-[trans-4-[2-[4-(3-chloro-benzo[d]isoxazol)-6-piperidinyl]ethyl]cyclohe-
xyl]-N,N-dimethylurea (compound 12);
##STR00019##
N'-[trans-4-[2-[4-(3-methyl-benzo[d]isoxazol)-6-piperazinyl]ethyl]cyclohe-
xyl]-N,N-dimethylurea (compound 13);
##STR00020##
N'-[trans-4-[2-[4-(6-methyl-benzo[d]isoxazol)-4-piperazinyl]ethyl]cyclohe-
xyl]-N,N-dimethylurea (compound 14).
##STR00021##
On the other hand, the present invention provides a method for
preparing the cyclohexane derivative or stereoisomer or salt
thereof, which comprises following steps:
reacting 4-ethylcyclohexylamine derivative II with
N,N-dimethylcarbamyl chloride III in the presence of an
acid-binding reagent to produce the compound IB:
##STR00022## wherein R and X are as defined above.
The molar ratio of the compound II to the compound III is 1-1.5:1,
the reaction temperature is 0.degree. C.-50.degree. C., the
acid-binding agent is an organic base selected from one or more of
triethylamine, diisopropylethylamine or pyridine, or an inorganic
base selected from one or more of sodium carbonate, potassium
carbonate, sodium bicarbonate or potassium bicarbonate; and the
molar ratio of the acid-binding agent (alkaline substance) to the
compound II is 1-1.5:1.
The present invention further provides a method for preparing the
compound II, which comprises following steps:
##STR00023## wherein R and X are as defined above, and Pg is an
amino-protecting group selected from benzyl (Bn), benzyl formate
(CBz) or t-butyloxycarbonyl (Boc). The compound of formula II may
be cis- or trans-; optionally, the cis- or trans-stereoisomers may
be obtained by subjecting the compound of formula IB to
chromatography separating or crystallizing. i) when X is N,
coupling piperazine with bromide to produce an intermediate IV;
wherein the coupling reaction is carried out in the presences of
palladium catalyst and a strong alkali selected from potassium
tert-butoxide, sodium tert-butoxide, potassium carbonate or cesium
carbonate, the reaction temperature is 50-150.degree. C., and the
molar ratio of piperazine to the bromide is 1-5:1; or when X is C,
the intermediate IV is commercially available (Shanghai Excellent
Chemical Co, Ltd.). ii) subjecting the intermediate IV and
4-amino-protected cyclohexaneacetaldehyde to a condensation and
imine reduction reaction to produce an intermediate V; wherein a
reducing agent used is a boron compound such as sodium
triacetoxyborohydride or sodium borohydride; the feeding molar
ratio of the intermediate IV to the 4-amino-protected
cyclohexaneacetaldehyde is 1-1.2:1; the molar ratio of the boron
compound to the intermediate IV is 1-2:1; and iii) removing the
amino-protecting group; in more particular, when Pg is t-butyloxy
carbonyl (Boc), removing the amino-protecting group by using an
acid which is a hydrogen chloride organic solution, or
trifluoroacetic acid, etc.; or when Pg is benzyl (Bn) or benzyl
formate (CBz), removing the amino-protecting group by hydrogenation
using palladium-on-carbon catalyst at a hydrogenation pressure of
0.1-1 Mpa.
The present invention also provides a use of the cyclohexane
derivatives or stereoisomers or salts thereof in preparing a drug
against neuropsychiatric diseases.
The cyclohexane derivatives or stereoisomers or salts thereof
according to the present invention are new compounds. These
compounds are produced with pharmacophore fusion-based drug design;
particularly, pharmacophore which potentially acts on the dopamine
D.sub.3 receptor and pharmacophore that has a potential inhibition
on 5-hydroxytryptamine absorption are fused, and then the compounds
are structurally modified and prepared, followed by in vitro test
on biological activity and in vivo test on anti-schizophrenic
activity, study on structure-activity relationship and
optimization. The pharmacological results show that the cyclohexane
derivatives or stereoisomers or salts thereof according to the
present invention have higher affinity to the D.sub.3 receptor and
5-hydroxytryptamine, but have lower affinity to the D.sub.2
receptor, demonstrating a high selectivity for D.sub.3/D.sub.2
receptors, and achieving an unexpected effect. The in vitro
receptor binding assay indicates that most of the cyclohexane
derivatives or stereoisomers or salts thereof show strong affinity
(Ki<10 nmol) to dopamine D.sub.3 receptor and 5-HT.sub.1A
receptor and weak affinity to dopamine D.sub.2 receptor (Ki>50
nmol), demonstrating excellent selectivity for D.sub.3/D.sub.2
receptors and strong affinity to 5-HT.sub.1A receptor. The in vivo
test on anti-schizophrenic activity shows that the cyclohexane
derivatives or stereoisomers or salts thereof function well on
schizophrenia. The study on structure-activity relationship shows
that the strong affinity to D.sub.3/D.sub.2 and 5-HT.sub.1A
receptors and high selectivity to D.sub.3/D.sub.2 receptors, as
well as the anti-schizophrenia effect are closely linked to the
benzoheterocycle fragments such as benzothiophene, benzisothiazole
or benzisoxazole in a series of cyclohexane derivatives according
to the present invention (in the prior art the corresponding
fragment of Cariprazine is 2,3-dichlorobenzene).
Therefore, the cyclohexane derivatives or stereoisomers or salts
thereof according to the present invention can be used in preparing
a drug against neuropsychiatric diseases.
The drug of the present invention is a pharmaceutical composition
consisting of the cyclohexane derivative or stereoisomer or salt
thereof as an active ingredient and a pharmaceutical excipient.
The pharmaceutical composition of the present invention may be
administrated in any convenient manner, for example, oral,
gastrointestinal, buccal, sublingual, nasal, rectal or transdermal.
The pharmaceutical composition may be made into different forms
such as solid and liquid preparations, for example suspensions,
tablets and capsules.
The composition in the form of a tablet may include fillers,
lubricants, adhesives and disintegrants conventionally used in
preparations. Liquid preparations are prepared by using a suitable
liquid carrier, such as a water-soluble solvent, e.g., water,
ethanol or glycerol, or a water-insoluble solvent e.g.,
polyethylene glycol, or a suspension or solution in the oil.
Particularly, the pharmaceutical composition is a solid tablet
consisting of the cyclohexane derivative of formula IB or formula I
or stereoisomer or salt thereof as an active ingredient and a
pharmaceutical excipient.
The solid tablet consists of the following components based on
weight: active ingredient: 1-40 mg diluent: 10-200 mg adhesive:
5-25 mg disintegrant: 5-50 mg and lubricant: 1-5 mg; wherein the
diluent is selected from starch, lactose or microcrystalline
cellulose; the adhesive is selected from hydroxybenzyl cellulose,
hydroxypropylmethyl cellulose or polyvinylpyrrolidone; the
disintegrant is selected from sodium hydroxyethyl starch or
crospovidone; and the lubricant is magnesium stearate.
Preferably, the pharmaceutical composition is a solid tablet
consisting of
N'-[trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]--
N,N-dimethylurea as an active ingredient and a pharmaceutical
excipient.
The solid tablet consists of the following components based on
weight: active ingredient: 1-40 mg diluent: 10-200 mg adhesive:
5-25 mg disintegrant: 5-50 mg and lubricant: 1-5 mg; wherein the
diluent is selected from starch, lactose or microcrystalline
cellulose; the adhesive is selected from hydroxybenzyl cellulose,
hydroxypropylmethyl cellulose or polyvinylpyrrolidone; the
disintegrant is selected from sodium hydroxyethyl starch or
crospovidone; and the lubricant is magnesium stearate.
Alternatively, the pharmaceutical composition is a suspension
consisting of the cyclohexane derivative of formula IB or formula I
or stereoisomer or salt thereof as an active ingredient and a
pharmaceutical excipient.
The suspension consists of the following components based on
weight: active ingredient: 1-40 mg diluent: 10-200 mg suspending
agent: 0.1-10 mg preservative: 0.01-0.5 mg buffer: 4-8 mg
co-solvent: 0-50 mg flavoring agent: 0-1 mg and colorant: 0-0.1 mg;
wherein the suspending agent is selected from xanthan gum or
microcrystalline cellulose; the preservative is selected from
sodium benzoate, methyl or ethyl p-hydroxybenzoate; the diluent is
selected from water or sorbitol; the buffer is citrate; the
co-solvent is selected from cyclodextrin, ethanol, propylene glycol
or polyethylene glycol; the flavoring agent can be a sweetener
known to the person skilled in the art (e.g., sugar or saccharin,
etc.); the colorant can be a fat-soluble or water-soluble colorant,
such as carotene, cocoa pigment or caramel pigment, etc.
Preferably, the present invention provides a suspension consisting
of
N'-[trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea as an active ingredient and a pharmaceutical
excipient.
The suspension consists of the following components based on
weight: active ingredient: 1-40 mg diluent: 10-200 mg suspending
agent: 0.1-10 mg preservative: 0.01-0.5 mg buffer: 4-8 mg
co-solvent: 0-50 mg flavoring agent: 0-1 mg and colorant: 0-0.1 mg;
wherein the suspending agent is selected from xanthan gum or
microcrystalline cellulose; the preservative is selected from
sodium benzoate, methyl or ethyl p-hydroxybenzoate; the diluent is
selected from water or sorbitol; the buffer is citrate; the
co-solvent is selected from cyclodextrin, ethanol, propylene glycol
or polyethylene glycol; the flavoring agent can be a sweetener
known to the person skilled in the art (e.g., sugar or saccharin,
etc.); the colorant is a fat-soluble or water-soluble colorant,
such as carotene, cocoa pigment or caramel pigment, etc.
The present invention further provides a use of the cyclohexane
derivatives or stereoisomers or salts thereof in preparing a drug
against neuropsychiatric diseases; and the use refers to the use of
the cyclohexane derivatives or stereoisomers or salts thereof in
preparing a drug for improving/treating schizophrenia, mental
disorders, dysphrenia, mental confusion, mood disorders, bipolar
disorders, depression, phobia, obsessive-compulsive disorders,
anxiety and cognitive disorders.
An acute toxicity experiment shows that the cyclohexane derivatives
of the present invention or stereoisomers or salts thereof have
very low toxicity (LD.sub.50>1,000 mg/Kg), which are
significantly superior to Cariprazine (LD.sub.50=75.3 mg/Kg),
indicating that the drug of the present invention is less toxic and
safe.
A pharmacological test shows that the cyclohexane derivatives of
the present invention or stereoisomers or salts thereof are novel
therapeutic drugs against neuropsychiatric disorders and have good
prospects on clinical application, which would be good news for
patients and bring good social benefits. In addition, the method
for preparing the compound of the present invention is simple and
easy to operate, suitable for industrial production and
valuable.
DETAILED DESCRIPTION OF THE INVENTION
Materials and agents used in the following examples are
commercially available, unless otherwise specified.
EXAMPLE 1
Preparation of 1-benzo[b]thiophene-4-piperazine hydrochloride
##STR00024##
A mixture of 7.20 g of 4-bromobenzo[b]thiophene, 19.9 g of
piperazine anhydride, 4.70 g of sodium tert-butoxide, 0.32 g of
(R)-(+)-2,2'-bis(diphenylphosphino)-1,1'-dinaphthalene (BINAP),
0.63 g of tris(dibenzylideneacetone)dipalladium and 150 ml of
toluene was refluxed at nitrogen atmosphere for 1 hour. 150 ml of
water was added to the mixture, extracted with 100 ml.times.3 of
ethyl acetate, washed with water, dried with magnesium sulfate and
then evaporated under reduced pressure to remove the solvent (0.01
MPa, 45.degree. C.). The remainder was purified by silica gel
column chromatography (methylene chloride:methanol:25% aqueous
ammonia=100:10:1) to obtain 4.60 g of
1-benzo[b]thiophen-4-yl-piperazine as a yellow oil. 2 ml of
concentrated hydrochloric acid was added to a methanol solution (25
ml) containing 4.6 g of 1-benzo[b]thiophen-4-yl-piperazine, and
evaporated under reduced pressure (0.01 MPa, 45.degree. C.) to
remove the solvent. Ethyl acetate (50 ml) was added to the
remainder to precipitate and crystallize. The resultant was
filtered and then dissolved in 15 ml of methanol under reflux.
After cooling to room temperature (25.degree. C.),
recrystallization was carried out to give the crystallized
1-benzo[b]thiophene-4-yl-piperazine hydrochloride as colorless
needles.
EXAMPLE 2
Preparation of
trans-4-[2-[4-(benzo[b]thiophen)-7-piperazinyl]ethyl]cyclohexyl-tert-buty-
l carbamate
##STR00025##
2.54 g (10 mmol) of 1-benzo[b]thiophene-4-piperazine hydrochloride
prepared in Example 1 and 2.40 g (10 mmol) of
trans-2-{1-[4-(N-tert-butoxycarbony)amino]cyclohexyl}-acetaldehyde
were dissolved in 120 ml of dichloromethane. 1.40 ml (10 mmol) of
triethylamine was added slowly at room temperature (25.degree.
C..+-.2.degree. C.), stirred for 10 minutes, and then 3.16 g (14.8
mmol) of sodium triacetoxyborohydride was added gradually. The
mixture was stirred at room temperature for reaction for 24 hours.
After the reaction was completed, 120 ml of 10% sodium bicarbonate
solution was added. The reaction system was directly extracted and
separated, the organic phase was dried with anhydrous sodium
sulfate, and filtered and evaporated to dryness. The solid was
refluxed to dissolve with 15 ml of ethyl acetate, and then cooled
to room temperature (25.degree. C..+-.2.degree. C.), crystallized
to give 3.70 g of the desired product.
.sup.1H-NMR (CDCl.sub.3) .delta. ppm: 7.81 (1H, brs), 7.78 (1H, d,
J=5.5 Hz), 7.73 (1H, d, J=8.1 Hz), 7.41 (1H, m), 7.30 (1H, d, 7.6
Hz), 6.94 (1H, d, J=7.6 Hz), 3.54 (1H, m), 3.35-3.23 (8H, m), 2.46
(2H, m), 1.86-1.65 (8H, m), 1.51-1.49 (1H, m), 1.42 (9H, s),
1.37-1.35 (2H, m).
EXAMPLE 3
Preparation of
trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexylamine
##STR00026##
4.43 g of
trans-4-[2-[4-(benzo[b]thiophen)-7-piperazinyl]ethyl]cyclohexyl-
-tert-butyl carbamate prepared in Example 2 was placed in a
reaction flask under an ice-water bath. 80 ml of a saturated
solution of hydrogen chloride in ethyl acetate was added to the
reaction flask. The reaction was stirred for 8 hours for
de-protection and finally a white precipitate was formed to give
3.42 g of the hydrochloride of the desired product. The resulting
solid was added to 50 ml of dichloromethane solution, and then 50
ml of a saturated solution of sodium bicarbonate was added, stirred
for 0.5 hour. This mixture was extracted and separated and the
organic phase was concentrated (0.01 MPa, 40.degree. C.) to give
3.30 g of the desired product.
.sup.1H-NMR (CDCl.sub.3) .delta. ppm: 7.78 (1H, d, J=5.5 Hz), 7.76
(1H, d, J=8.1 Hz), 7.37 (1H, m), 7.29 (1H, d, 7.6 Hz), 6.96 (1H, d,
J=7.6 Hz), 3.48-3.38 (8H, m), 2.53 (1H, m), 2.46 (2H, m), 1.78-1.63
(8H, m), 1.51-1.49 (1H, m), 1.42 (2H, brs), 1.37-1.35 (2H, m).
EXAMPLE 4
Preparation of
N'-[trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea (Compound 1)
##STR00027##
1.73 g of
trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexy-
lamine prepared in Example 3 was dissolved in 50 ml of
dichloromethane. 1.40 ml of triethylamine was added, and then 5.50
mmol of N,N-dimethylcarbamyl chloride was added. The mixture was
stirring for 48 hours at room temperature (25.degree.
C..+-.2.degree. C.). After the reaction was completed, 50 ml of
water was added to extract and separate the reactant. The organic
phase was concentrated (0.01 MPa, 45.degree. C.) and the desired
component was collected by using column chromatography of
methanol:dichloromethane=1:10 (400 mesh silica gel) and then
concentrated to give 1.89 g of the amorphous desired product.
.sup.1H-NMR (CDCl.sub.3) .delta. ppm: 7.79 (1H, d, J=5.5 Hz), 7.76
(1H, d, J=8.1 Hz), 7.33 (1H, m), 7.28 (1H, d, 7.6 Hz), 6.96 (1H, d,
J=7.6 Hz), 6.48 (1H, brs), 3.44-3.36 (8H, m), 3.58 (1H, m), 3.01
(6H, s), 2.46 (2H, m), 1.68-1.42 (8H, m), 1.52-1.48 (1H, m),
1.38-1.36 (2H, m).
EXAMPLE 5
Preparation of
N'-[trans-4-[2-[7-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea (Compound 2)
Compound 2 was prepared according to the procedures given in
Examples 1-4 by using 7-bromobenzo[b]thiophene as a starting
material.
.sup.1H-NMR (CDCl.sub.3) .delta. ppm: 7.78 (1H, d, J=5.6 Hz), 7.76
(1H, d, J=8.0 Hz), 7.31 (1H, m), 7.27 (1H, d, 7.6 Hz), 6.98 (1H, d,
J=7.2 Hz), 6.44 (1H, brs), 3.48-3.42 (8H, m), 3.54 (1H, m), 3.00
(6H, s), 2.46 (2H, m), 1.68-1.42 (8H, m), 1.52-1.48 (1H, m),
1.38-1.36 (2H, m).
EXAMPLE 6
Preparation of
N'-[trans-4-[2-[4-(benzo[c]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea (Compound 3)
Compound 3 was prepared according to the procedures given in
Examples 1-4 by using 4-bromobenzo[c]thiophene as a starting
material.
.sup.1H-NMR (CDCl.sub.3) .delta. ppm: 7.33 (2H, s), 7.27-7.25 (1H,
m), 7.27 (1H, d, 7.6 Hz), 6.77 (1H, d, J=7.2 Hz), 6.73 (1H, d,
J=7.2 Hz), 6.44 (1H, brs), 3.48-3.42 (8H, m), 3.54 (1H, m), 2.99
(6H, s), 2.46 (2H, m), 1.68-1.42 (8H, m), 1.52-1.48 (1H, m),
1.46-1.42 (2H, m).
EXAMPLE 7
Preparation of
N'-[trans-4-[2-[4-(6-fluoro-benzo[d]isoxazol)-3-piperazinyl]ethyl]cyclohe-
xyl]-N,N-dimethylurea (compound 5)
Compound 5 was prepared according to the procedures given in
Examples 1-4 by using 6-fluoro-3-bromo-1,2 benzo[d]isoxazole as a
starting material.
.sup.1H-NMR (CDCl.sub.3) .delta. ppm: 7.41 (1H, d), 7.12 (1H, d),
6.98 (1H, s), 6.52 (1H, brs), 3.55 (1H, m), 3.46-3.42 (8H, m), 2.99
(6H, s), 2.45 (2H, m), 1.68-1.40 (8H, m), 1.50-1.48 (1H, m),
1.37-1.35 (2H, m).
EXAMPLE 8
Preparation of
N'-[trans-4-[2-[4-(3-chloro-benzo[d]isoxazol)-6-piperazinyl]ethyl]cyclohe-
xyl]-N,N-dimethylurea (Compound 6)
Compound 6 was prepared according to the procedures given in
Examples 1-4 by using 3-chloro-6-bromo-benzo[d]isoxazole as a
starting material.
.sup.1H-NMR (CDCl.sub.3) .delta. ppm: 7.25 (1H, d), 6.78 (1H, s),
6.72 (1H, d), 6.51 (1H, brs), 3.54 (1H, m), 3.46-3.42 (8H, m), 2.99
(6H, s), 2.45 (2H, m), 1.67-1.40 (8H, m), 1.50-1.48 (1H, m),
1.42-1.35 (2H, m).
EXAMPLE 9
Preparation of
N'-[trans-4-[2-[4-(6-fluoro-benzo[d]isoxazol)-3-piperidyl]ethyl]cyclohexy-
l]-N,N-dimethylurea (Compound 7)
##STR00028##
Compound 7 was prepared according to the procedures given in
Examples 2-4 by using 6-fluoro-3-piperidin-4-yl-1,2
benzo[d]isoxazole (available from Shanghai Excellent Chemical Co.,
Ltd.) as a starting material.
.sup.1H-NMR (CDCl.sub.3) .delta. ppm: 7.41 (1H, d), 7.12 (1H, d),
6.97 (1H, s), 6.51 (1H, brs), 3.55-3.53 (1H, m), 2.99 (6H, s),
2.78-2.76 (1H, m), 2.66-2.37 (4H, m), 2.46-2.40 (2H, m), 1.78-1.68
(12H, m), 1.50-1.48 (1H, m), 1.37-1.34 (2H, m).
EXAMPLE 10
Preparation of solid tablets of
N'-[trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea (Compound 1)
1000 tablets each weighing 200 mg were prepared according to the
formulation shown in the table below.
TABLE-US-00001 Amount Composition (g) Compound 1(active ingredient;
20.0 prepared in Example 4) Lactose 126.0 Microcrystalline
Cellulose 42.0 Hydroxypropylmethyl Cellulose 4.0 Sodium
Hydroxyethyl Starch 6.0 Magnesium Stearate 2.0
The preparation method comprising: the active ingredient, lactose,
microcrystalline cellulose and sodium hydroxyethyl starch were
mixed and added to a high-shear wet granulator, stirring well at a
certain rotating speed. Afterwards, 50.0 g of an aqueous solution
of hydroxypropylmethylcellulose was added to the mixture to make it
into appropriate granules under high-speed shearing conditions. The
wet granules then were dried over a fluidized bed, and the
resulting dried granules were uniformly mixed with the magnesium
stearate and then compressed into tablets.
EXAMPLE 11
Preparation of an oral suspension of
N'-[trans-4-[2-[4-(benzo[b]thiophene)-7-piperazinyl]ethyl]cyclohexyl]-N,N-
-dimethylurea (Compound 1)
1000 bottles each containing 5 ml (10 mg/ml as per specification)
were prepared according to the formulation shown in the table
below.
TABLE-US-00002 Amount Composition (g) Compound 1(active ingredient;
50.0 prepared in Example 4) water 3500.0 Polyethylene Glycol 50.0
Sorbitol 500.0 Microcrystalline Cellulose 25.0 Xanthan Gum 5.0
Methyl p-Hydroxybenzoate 1.25 Ethyl p-Hydroxybenzoate 1.25 Citrate
Adjusting pH to 4-8
The preparation method comprising: methyl p-hydroxybenzoate and
ethyl p-hydroxybenzoate were dissolved in hot water and then cooled
to room temperature (25.degree. C..+-.2.degree. C.). Then,
sorbitol, polyethylene glycol, xanthan gum, citrate, the compound 1
with an average particle size of 30 .mu.m and microcrystalline
cellulose were successively added, and stirred well to give an oral
suspension.
EXAMPLE 12
Pharmacological Test
I. In Vitro Test on Biological Activity of the Cyclohexane
Derivative of the Present Invention.
This test was carried out according to the dopamine D.sub.2/D.sub.3
receptor binding assay and the 5-HT.sub.1A receptor binding assay
described in Jiangsu Hengyi Pharmaceutical Co., Ltd.'s Patent CN
103130737 A. IC50 values were calculated from
concentration-dependent reactions using a non-linear analysis
program. Ki values were calculated from IC50 values by using
Cheng-Prussoff equation, i.e. Ki=IC.sub.50/(1+[L]/KD), wherein Ki
is the affinity of the drug to the receptor; L is the concentration
of the compound to be tested; and KD is the affinity of radioligand
to the receptor.
(I) Dopamine D.sub.2 Receptor Binding Assay
i. Materials
a. Transfection of D.sub.2 Receptor Cells: by using calcium
phosphate method, HEK293 cells were transfected with a plasmid
vector containing the D.sub.2 receptor protein gene. The
transfected cells were cultured in a culture solution containing
G418, followed by a selection of monoclonal cell and radioligand
binding assay, finally obtaining a stable cell line with stable
expression of D.sub.2 receptor protein. b. Materials for receptor
binding assay: isotope ligand [.sup.3H] Spiperone (113.0 Ci/mmol);
available from Sigma-Aldrich Company; (+) spiperone, available from
RBI company; GF/B glass fiber filter paper, available from Whatman
Company; Tris imported and packaged; PPO, POPOP, available from
Shanghai Reagent Chemical Company; lipid-soluble scintillation
solution; and Beckman LS-6500 Multi-function Liquid Scintillation
Counter. ii. Test Method a. Treatment of cells: HEK-293 cells were
infected with various recombinant viruses of the above genes,
respectively, and the receptor proteins were highly expressed on
the membranes after 48-72 hours. The cells were centrifuged at 1000
rpm for 5 minutes. The culture solution was discarded, and the
cells were collected and stored in a refrigerator at -20.degree. C.
for standby. The cells were resuspended with Tris-HCl buffer
(pH=7.5) when tested. b. Competitive binding assay for receptor
The compound to be tested and the radio ligand, both 20 .mu.L, and
160 .mu.L of receptor proteins were added to the reaction test
tubes to achieve a final concentration of 10 .mu.mol/L for each of
the compound to be tested and positive control, incubated in a
water bath of 30.degree. C. for 50 minutes and immediately
transferred to an ice bath to terminate the reaction; placed on a
Millipore cell sample collector, filtered by suction rapidly
through a GF/C glass fiber filter, and eluted with 3 mL of eluent
(50 mM Tris-HCl, pH7.5) for three times and dried with microwave
for 5-6 minutes. The filter paper was transferred into a 0.5 mL
centrifuge tube to which 500 .mu.L of lipid-soluble scintillation
solution was added, and settled away from light for more than 30
minutes. The radioactive intensity was determined by counting. The
concentration of the compound was 10 .mu.mol/L, and inhibition rate
(%) of each compound to the binding of isotope ligands was
calculated according to the following equation: Inhibition rate
(I%)=(total binding tube cpm-compound cpm)/(total binding tube
cpm--non-specific binding tube cpm).times.100%. (II) Dopamine
D.sub.3 Receptor Binding Assay
The concentration of the compound was 10 .mu.mol/L, and the assay
was performed according to the method described in Journal of
Pharmacology and Experimental Therapeutics 2010, 333 (1): 328.
(III) 5-HT.sub.1A Receptor Binding Assay
i. Materials
5-HT.sub.1A receptor isotope ligand [.sup.3H] 8-OH-DPAT (available
from PE Company); (+)5-hydroxytrptamine (available from Sigma
Company); GF/B glass fiber filter paper (available from Whatman
Company); lipid-soluble scintillation solution: PPO, POPOP
(available from Shanghai Chemical Reagent Company); toluene (from
Sinopharm Chemical Reagent Co., Ltd.); Tris imported and
packaged.
Treatment of cells: HEK-293 cells which stably express the
5-HT.sub.1A receptor by gene recombination were cultured in
DMEM+10% serum solution for 3-5 days and then collected with PBS.
The cells were centrifuged at 3000 rpm and -4.degree. C. for 10
minutes. Afterwards, the supernate was discarded, and the cells
were collected and stored in a refrigerator at -80.degree. C. The
cells were resuspended with D.sub.1 Binding Buffer (pH 7.4) when
tested.
ii. Test Method
Inhibition rate of each compound of 10 umol/L to the binding of
[.sup.3H]8-OH-DPAT and the 5-HT.sub.1A receptor was determined by a
general selection.
The experimental data is shown in Table 1.
TABLE-US-00003 TABLE 1 Binding Assay of Compounds to D.sub.2 and
D.sub.3 Receptors and Affinity to 5-HT.sub.1A Receptor (Ki: nmol)
Compounds D.sub.2 D.sub.3 5-HT.sub.1A ##STR00029## >1000 0.06
3.85 ##STR00030## 978.2 0.07 3.72 ##STR00031## >1000 0.06 3.96
##STR00032## 1.3 0.29 4.12 ##STR00033## 250.9 0.57 22.83
##STR00034## >1000 0.15 3.65
Conclusion: it can be seen from the experimental results in Table 1
that the series of compounds of the present invention have a strong
affinity to D.sub.3 and a very weak affinity to D.sub.2 with nearly
ten thousand folds difference between them, demonstrating that the
series of compounds have high selectivity to D.sub.2/D.sub.3
receptors and reduce side effects when selecting D.sub.2 receptor.
Further, the compounds show relatively strong affinity to
5-HT.sub.1A receptor and act on a wide spectrum of neuropsychiatric
diseases.
EXAMPLE 13
In Vivo Anti-Schizophrenic Activity Assay of the Cyclohexane
Derivatives of the Present Invention
I. MK-801 Model
(I) Modeling of MK-801-Induced Schizophrenia in Mice
100 Sprague-Dawley rats (provided by Shanghai Lake Experimental
Animal Co., Ltd.), all males, were randomly divided into 10 groups
according to body weights: blank control, MK-801 model control, the
cyclohexane derivatives C.sub.1 to C.sub.8 (corresponding to the
compound 1 to the compound 7 and compound 14; 0.3 mg/kg) and
Cariprazine positive control (prepared according to the method
described in Patent CN103130737A; 0.3 mg/kg). Each rat was placed
in a soundproof box for 30 minutes on the day before the test to
adapt. On the next day, each rat was administrated with the
respective test compounds, and after 30 minutes, administrated
intraperitoneally with a 0.3 mg/kg MK-801 solution at 5.0 mL/kg
body weight of the rat to build a model of MK-801-induced
schizophrenia in mice.
Administration: the rats in the present cyclohexane derivatives and
the cariprazine positive control groups were orally administered
(i.g.), while the MK-801 model control group was administered
intraperitoneally.
(II) Observation of Open Field Running Behavior
Mice were administered with MK-801 and then immediately placed in
the soundproof box. The total distances of motion of mice within
2.5 hours were observed and recorded. Improvement rate=(total
distance of model control-total distance of administration)/(total
distance of model control).times.100%. (III) Statistical Method
All the data was expressed with x.+-.SD and processed by SPSS17.0
software statistical package. T-test and one-way analysis of
variance were performed to compare the mean of two samples, with
P<0.05 as significant difference.
(IV) Results
The results specifically are shown in Table 2 below.
TABLE-US-00004 TABLE 2 Effect of Single Oral Administration of the
Cyclohexane Derivatives on the Total Distance of Open Field Motion
in MK-801-Induced Schizophrenia in Mice Model (x .+-. SD) Total
Distance Improve- Num- within 61-150 ment ber of Dosage minutes
Rate Groups Rats (mg/kg) (m) (%) Blank Control 10 -- -- MK-801Model
10 0.3 mg/kg 309.78 .+-. 39.1 -- Control Cariprazine 10 0.3 mg/kg
168.25 .+-. 26.9**** 45.69 Positive Control C1 (Compound 1) 10 0.3
mg/kg 73.09 .+-. 11.7**** 76.41 C2 (Compound 2) 10 0.3 mg/kg 46.95
.+-. 10.3**** 84.84 C3 (Compound 3) 10 0.3 mg/kg 63.79 .+-.
10.8**** 79.41 C4 (Compound 4) 10 0.3 mg/kg 232.18 .+-. 20.3* 25.05
CS (Compound 5) 10 0.3 mg/kg 5.43 .+-. 1.7**** 98.25 C6 (Compound
6) 10 0.3 mg/kg 171.78 .+-. 13.0* 12.27 C7 (Compound 7) 10 0.3
mg/kg 160.82 .+-. 12.91* 48.09 C8 (Compound 14) 10 0.3 mg/kg 150.28
.+-. 11.70* 51.49
The results in Table 2 show that the cariprazine positive control
and the cyclohexane derivatives (Compound 1 to Compound 7) of the
present invention have decreased the total distances of the rats
within 150 minutes as compared with the MK-801 model control.
Wherein *P<0.05, ****P<0.0001.
These results demonstrate that: i. compared with the blank control,
the distance of the open-field motion of MK-801 model control has
increased significantly, indicating that MK-801 may causes
schizophrenia in mice; ii. compared with the MK-801 model control,
and the cariprazine positive control has significantly inhibited
the high spontaneous activity of MK-801-induced rats (P <0.0001)
at a dose of 0.3 mg/kg; the cyclohexane derivatives, Compounds 1,
2, 3 and 5 of the present invention can significantly inhibit the
high spontaneous activity of MK-801-induced rats (P<0.0001) at a
dose of 0.3 mg/kg, and the cyclohexane derivatives, Compounds 6, 7
and 14 of the present invention also have significantly inhibited
the high spontaneous activity of MK-801-induced rats (P<0.05) at
a dose of 0.3 mg/kg , which are comparable with the cariprazine
positive control. Since the MK-801-induced open-field motion model
is closely related to the symptoms of schizophrenia, it is
indicated that the cyclohexane derivative series of the present
invention have a significant effect on schizophrenia.
EXAMPLE 14
Acute Toxicity Assay of the Cyclohexane Derivatives of the Present
Invention
ICR mice orally administered with the compounds of the present
invention were evaluated and their toxicity symptoms after oral
administration were observed, as well as the mortality, which was
calculated by Bliss method, thus the acute toxicities were
compared.
Experimental Program
i. Preparation of solvent: Tween-80 of an appropriate amount was
diluted with deionized water to a concentration of 5% (g/v)
Tween-80. ii. Formulations for administration: test samples were
weighed as required, respectively, and made into suspensions with
5% the Tween-80 solution to achieve concentrations of 0.94, 1.88,
3.75, 7.5, 15, 30 and 60 mg/mL, respectively (equivalent to 18.75,
37.5, 150, 300, 600 and 1200 mg/kg, respectively). iii.
Administration: the test compound and solvent medium control groups
(0.5% Tween-80) were administered orally.
Observation of general symptoms: Day 1: rats were observed at about
10 minutes, 0.5, 2, 4 and 6 hours after the first administration,
respectively; Days 2-6, mice were observed twice a day, once in the
morning and once in the afternoon. Observations include but are not
limited to: general condition, behavior, gait, eyes, mouth, nose,
gastrointestinal tract, skin and hair and urogenital tract.
The experimental results are shown in Table 4.
TABLE-US-00005 TABLE 4 Acute Toxicity of Single Oral Administration
of Compounds Compounds LD.sub.50 (mg/Kg) ##STR00035## 1100 mg/KG
##STR00036## 1050 mg/KG ##STR00037## 1080 mg/KG
Conclusion: the acute toxicity of the cyclohexane derivatives of
the present invention is far lower than that of the cariprazine
positive control (RGH-188, 75.3 mg/kg), showing good safety.
* * * * *